Doctor of Philosophy (Ph.D.)
Degree Granting Department
Biology (Cell Biology, Microbiology, Molecular Biology)
Meera Nanjundan, Ph.D.
Brant Burkhardt, Ph.D.
Kristina Schmidt, Ph.D.
Sandy Westerheide, Ph.D.
EVI1, Fallopian Tube Secretory Epithelial Cells, Hydroxychloroquine, NCOA4
For women in the United States, ovarian cancer (OVCA) represents the deadliest gynecological cancer with over 14,000 deaths predicted to occur in 2019 . Although innovative treatment strategies have improved the 5-year outcome for these patients, the overall survival has not changed in over 40 years . This is largely due to the challenges in detecting this heterogenous disease at an early stage, as there are multiple potential precursors that may give rise to different histological subtypes . This dissertation is thus focused on investigating pro-survival pathways involved in the pathogenesis of endometriosis (a proposed precursor) and epithelial ovarian cancers.
Endometriosis occurs when glands (comprised of both epithelial and stromal cells) from the endometrium are located in the pelvic cavity . A cellular homeostasis process known as autophagy is characterized as being dysregulated in patients with endometriosis [16-20], but the contribution of this pathway to the formation of endometriotic lesions has been uncertain. Furthermore, studies have identified that patients diagnosed with endometriosis also have an increased risk for developing more rare epithelial OVCA (EOC), namely endometrioid and clear cell OVCAs [21, 22], which suggests that endometriosis is a likely precursor. In Chapter 2 of this dissertation, we characterized the mRNA and protein expression of autophagy markers in endometriosis-induced mice and from patient specimens. We further identified that inhibiting the autophagy pathway with an FDA-approved anti-malarial drug, hydroxychloroquine (HCQ), significantly reduced human endometriotic cell viability in vitro and reduced the number of endometriotic lesions that developed within an in vivo endometriosis model. These results suggest that autophagy may be an important contributor to the establishment of endometriosis.
EOCs are characterized by genomic aberrations which can help in distinguishing the different histological subtypes [23-25]. Furthermore, recent studies suggest that iron dependency may be an important characteristic of OVCA , but further research into the contribution of iron dysregulation to ovarian cancer pathogenesis is needed. Thus, in Chapter 3, we generated transformed human endometriotic cell lines by inhibiting p53 (which is commonly inactivated via mutations in ~95% of high-grade serous OVCA (HGSOC) ) and overexpressing human telomerase reverse transcriptase (hTERT, which is commonly amplified in HGSOC ). These cells were also expressing oncogenic c-Myc (which is amplified at 8q24 in HGSOC ) as well as mutated (constitutively activated) H-Ras (which is commonly activated in OVCA ). We characterized increased colony forming ability and reduced senescence compared to non-transformed control cells, as expected, but also identified mRNA expression changes in key regulators of the iron pathway. One such regulator, NCOA4 (nuclear receptor coactivator 4), promotes the release of iron from the ferritin storage complex through a targeted autophagy process, ferritinophagy [3, 30]. Interestingly, NCOA4 mRNA and protein expression were elevated in transformed endometriotic cells relative to controls. We further identified increased NCOA4 mRNA and protein in malignant OVCA cell lines relative to non-malignant cells, as well as increased NCOA4 protein in human OVCA tumors relative to control adjacent tissues. These findings suggest a potential role for NCOA4 and iron dysregulation in OVCA pathogenesis.
Iron is an essential metal required for many cellular processes [31-33], but it can also be detrimental through its ability to produce reactive oxygen species (ROS) [34, 35]. Excess iron has been implicated in the development of epithelial cancers, such as hepatocellular cancer . Recent evidence also supports a role for iron in ovarian cancer pathogenesis , but the molecular events mediating this process have not been identified. We thus investigated the contribution of iron to the transition of precursors to OVCA in Chapter 4 by chronically exposing fallopian tube secretory epithelial cells (FTSECs, another proposed precursor to OVCA ) to a source of non-transferrin bound iron (NTBI). In addition, we generated transformed FTSECs and compared the functional and expressional changes between the two cell lines. We found that both chronic iron exposure and cellular transformation increased cell numbers and observed altered protein and mRNA expression of MECOM genes, which are commonly aberrant in OVCA . Specifically, the expression of tumor suppressive MDS1/EVI1 was reduced while the expression of oncogenic EVI1 and EVI1Del190-515 was elevated in both conditions. Interestingly, we also observed differences between these cell lines, such as increased migratory capacity and elevated b-catenin protein expression with chronic iron treatment (but not with oncogenic transformation). Together, these findings demonstrate that iron may mediate the transition of fallopian tube precursors to a transformed-like state.
Altogether, these findings have improved our knowledge of pathways that may be involved in the pathogenesis of endometriosis and epithelial ovarian cancers, namely the autophagy and iron regulatory pathways. Additional research is needed, however, in order to identify the respective underlying mechanisms. Such studies would also expand our understanding of the contribution of iron to the transition of endometriotic and fallopian tube precursor cells to OVCA. Understanding these mechanisms will promote innovative strategies to detect these diseases early and help improve current treatment strategies, thus improving on the clinical outcome for women diagnosed with ovarian cancer.
Scholar Commons Citation
Rockfield, Stephanie, "Characterization of the Autophagic-Iron Axis in the Pathophysiology of Endometriosis and Epithelial Ovarian Cancers" (2019). Graduate Theses and Dissertations.